We have studied a serum activity that enhances in vitro ICAM-1 expression by human endothelial cells (EC) and report that this activity can be found in approximately 8% of pretransplant serum samples from individuals with a history of high %PRA. Hence, most high %PRA sera lack this activity, and, furthermore, mixing these negative sera does not result in an active serum pool. In patients with active serum, the ICAM-1 enhancing activity is found only sporadically, despite the continuous detection of endothelial-reactive antibodies. Absorption of Ig from a high %PRA serum reduced ICAM-1 enhancing activity, as well as endothelial-reactive antibodies. However, enhancing activity can sometimes be observed in sera that lack detectable endothelial-reactive antibodies, and none of several patient sera with defined MHC class I-specific alloantibodies displayed ICAM-1 enhancing activity. Together, these data suggest that ICAM-1 enhancing activity may not necessarily be mediated by anti-MHC alloantibodies. In addition to influencing this expression, ICAM-1 active patient sera also influence EC expression of VCAM-1 and MHC class I, but not MHC class II molecules, a pattern that is similar to that stimulated by TNF alpha. However, coincubation of EC with active serum plus soluble TNF receptor did not block the endothelial phenotypic changes, despite the ability of the soluble receptor to completely abrogate endothelial changes induced by TNF alpha. IFN gamma also increases endothelial ICAM-1 expression, but has response kinetics different from that of active serum. Interestingly, brief treatment of endothelial cells with IFN gamma greatly increased the amount of IgG bound from the active sera by EC. We conclude that some pretransplant patients occasionally express an activity in their serum that influences EC expression of several adhesion molecules, including ICAM-1, VCAM-1, and MHC class I. This activity may be associated with alloantibodies, but is independent of MHC class I-reactive antibodies, circulating TNF alpha, or IFN gamma. The relevance of a serum-borne component capable of activating EC is discussed.